Project Summary: Nuclear RNA surveillance is a fundamental quality assurance mechanism critical for the accurate expression of functional RNAs and the timely destruction of RNAs that are either regulatory in nature or byproducts of processing. Any imbalance that occurs in nuclear RNA surveillance has the potential to cause aberrant RNA production and accumulation, which can have pathological consequences. It is therefore important to develop a complete understanding of the nuclear RNA surveillance mechanism by studying its components. Incorrectly formed RNAs are polyadenylated by the TRAMP complex, composed of a polyA polymerase (Trf4p/Trf5p), RNA helicase (Mtr4p) and Zn-binding protein (Air1p/Air2p), and targeted for degradation by the nuclear exosome. Our understanding of what each TRAMP subunit contributes to RNA recognition, adenylation/modulation, structure alterations and communication with exosome is poorly developed despite its pivotal position in nuclear RNA surveillance. To overcome this and develop a more complete understanding of how TRAMP distinguishes defective from functional RNA, we will undertake to discover answers to basic questions about, how TRAMP works as a three member complex, how each subunit influences the other members in RNA recognition, modification, and communication with the degrading machinery, the nuclear exosome. This will be accomplished using established methodologies in genetics, and biochemical analyses of proteins, protein-complexes and RNA processing and degradation. Capitalizing on proven technology with a new purpose, we are developing a method to characterize loss of TRAMP function in cells with respect to adenylation and RNA unwinding.